U.S. patent number 7,316,906 [Application Number 10/211,394] was granted by the patent office on 2008-01-08 for cd38 as a prognostic indicator in b cell chronic lymphocytic leukemia.
This patent grant is currently assigned to The Feinstein Institute for Medical Research. Invention is credited to Nicholas Chiorazzi, Rajendra N. Damle, Tarun Wasil.
United States Patent |
7,316,906 |
Chiorazzi , et al. |
January 8, 2008 |
CD38 as a prognostic indicator in B cell chronic lymphocytic
leukemia
Abstract
The subject invention discloses a method for determining the
prognosis and probable clinical course of a subject diagnosed with
B-CLL. Specifically, the invention involves comparing CD38
expression in a biological sample from the subject containing B-CLL
cells to a baseline level of CD38 expression, wherein an elevated
level of CD38 expression in relation to the baseline level of CD38
expression may indicate poor prognosis or aggressive course of
disease in the subject. Also disclosed is a method for determining
whether the Ig V genes of the B-CLL cells of a B-CLL patient are
mutated, comprising comparing CD38 expression in a biological
sample from the subject containing B-CLL cells to a baseline level
of CD38 expression, wherein a lower level of CD38 expression in
relation to the baseline level indicates IG V gene mutation.
Inventors: |
Chiorazzi; Nicholas (Tenafly,
NJ), Damle; Rajendra N. (Lynbrook, NY), Wasil; Tarun
(St John's, CA) |
Assignee: |
The Feinstein Institute for Medical
Research (Manhasset, NY)
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Family
ID: |
23645518 |
Appl.
No.: |
10/211,394 |
Filed: |
August 2, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20030082593 A1 |
May 1, 2003 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09415393 |
Oct 8, 1999 |
6506551 |
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Current U.S.
Class: |
435/7.1; 435/4;
435/7.2; 435/7.21; 435/7.23 |
Current CPC
Class: |
C12Q
1/6886 (20130101); G01N 15/1456 (20130101); G01N
33/57426 (20130101); A61K 31/7076 (20130101); C12Q
2600/106 (20130101); C12Q 2600/118 (20130101); G01N
2015/149 (20130101); C12Q 2600/156 (20130101); C12Q
2600/158 (20130101) |
Current International
Class: |
G01N
33/53 (20060101); C12Q 1/00 (20060101); G01N
33/567 (20060101); G01N 33/574 (20060101) |
Field of
Search: |
;435/7.1 |
References Cited
[Referenced By]
U.S. Patent Documents
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6506551 |
January 2003 |
Chiorazzi et al. |
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Other References
Damle et al (Blood, 1999, 94:1840-1847). cited by examiner.
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Primary Examiner: Ungar; Susan
Attorney, Agent or Firm: Amster, Rothstein & Ebenstein
LLP
Government Interests
STATEMENT OF GOVERNMENT INTEREST
This invention is supported in part by US PHS grant AI 10811 from
the NIH NIAID. As such, the U.S. Government may have certain rights
in this invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This is a continuation of U.S. patent application Ser. No.
09/415,393, filed Oct. 8, 1999, now U.S. Pat. No. 6,506,551 B1.
Claims
What is claimed is:
1. A method for determining indications of the prognosis of and
aggressiveness of disease in a subject with B cell chronic
lymphocytic leukemia ("B-CLL"), the method comprising: measuring
CD38 expression by B-CLL cells in a blood sample from the subject;
determining the percentage of B-CLL cells in the sample that are
CD38.sup.+; and comparing said percentage to a cut-off value,
wherein a percentage of CD38.sup.+ B-CLL cells from the subject
that is greater than or equal to the cut-off value indicates a poor
prognosis and an aggressive disease course and a percentage of
CD38.sup.+ B-CLL cells from the subject that is less than the
cut-off value indicates an indolent disease course, wherein the
cut-off value is determined by: measuring CD38 expression by B-CLL
cells in a blood sample from each of a cohort of B-CLL patients;
determining the percentage of B-CLL cells that are CD38.sup.+ in
each of the cohort patients: determining whether the B-CLL in each
of the cohort patients is aggressive or indolent; and plotting the
percentage of B-CLL cells that are CD38.sup.+ in the cohort
patients vs. aggressiveness or indolence in the cohort patients to
establish a cut-off value that distinguishes between patients with
aggressive disease and patients with indolent disease.
2. The method of claim 1, wherein B-CLL cells are determined as
CD5.sup.+ and CD19.sup.+.
3. The method of claim 1, wherein CD38 expression of B-CLL cells is
determined using flow cytometry.
4. The method of claim 1, wherein the B-CLL patients are IgM.sup.+
B-CLL patients.
5. The method of claim 1, wherein measuring CD38 expression by
B-CLL cells in a blood sample from each cohort patient is
determined prior to measuring CD38expression by B-CLL cells in a
blood sample from the subject.
6. A method for evaluating whether a subject with B cell chronic
lymphocytic leukemia ("B-CLL") should undergo an aggressive course
of treatment, the method comprising measuring CD38 expression by
B-CLL cells in a blood sample from the subject; determining the
percentage of B-CLL cells in the sample that are CD38.sup.+; and
comparing said percentage to a cut-off value, wherein a percentage
of CD38.sup.+ B-CLL cells from the subject that is greater than or
equal to the cut-off value indicates the subject should undergo an
aggressive course of treatment and a percentage of CD38.sup.+ B-CLL
cells from the subject that is less than the cut-off value
indicates the subject should not undergo an aggressive course of
treatment, wherein the cut-off value is determined by: measuring
CD38 expression by B-CLL cells in a blood sample from each of a
cohort of B-CLL patients; determining the percentage of B-CLL cells
that are CD38.sup.+ in each of the cohort patients; determining
whether the B-CLL in each of the cohort patients is aggressive or
indolent; and plotting the percentage of B-CLL cells that are
CD38.sup.+ in the cohort patients vs. aggressiveness or indolence
in the cohort patients to establish a cut-off value that
distinguishes between patients with aggressive disease and patients
with indolent disease.
7. The method of claim 6, wherein B-CLL cells are determined as
CD5.sup.+ and CD19.sup.+.
8. The method of claim 6, wherein CD38 expression of B-CLL cells is
determined using flow cytometry.
9. The method of claim 6, wherein the B-CLL patients are IgM.sup.+
B-CLL patients.
10. The method of claim 6, wherein measuring CD38 expression by
B-CLL cells in a blood sample from each cohort patient is
determined prior to measuring CD38 expression by B-CLL cells in a
blood sample from the subject.
Description
BACKGROUND OF THE INVENTION
B cell chronic lymphocytic leukemia (B-CLL) is the most common
leukemia in the Western world (Rai K, Patel D: Chronic Lymphocytic
Leukemia, in Hoffman R, Benz E, Shattil S, Furie B, Cohen H,
Silberstein L (eds): Hematology: Basic Principles and Practice (ed
2nd). New York, Churchill Livingstone, 1995, p 1308). Around 7,500
individuals develop and 5,000 die from this disease each year
(Landis S H, et al., CA Cancer J Clin 48:6, 1998). Age is an
important factor, since the incidence of B-CLL increases linearly
with each decade above the age of 40 (Ries L, et al: SEER cancer
statistics review 1973-1991: Tables and graphs., in Ries L, et al
(eds). Bethesda, NIH, 1994; Rai K R, Clin Geriatr Med 13:245,
1997). In addition, gender is relevant, since men outnumber women
by an approximate 2:1 ratio (Catovsky D, et al., Br J Haematol
72:141, 1989) and may have a worse clinical outcome (Id.; Mandelli
F, et al., J Clin Oncol 5:398, 1987).
Patients with B-CLL follow heterogeneous clinical courses. Some
survive for prolonged periods without definitive therapy, while
others die rapidly despite aggressive treatment (Rai K, Patel D:
Chronic Lymphocytic Leukemia, in Hoffman R, et al. (eds):
Hematology: Basic Principles and Practice (ed 2nd). New York,
Churchill Livingstone, 1995, p 1308; Zwiebel J A, Cheson B D, Semin
Oncol 25:42, 1998). While various staging systems, most notably the
Rai and Binet staging systems, have been developed to address this
clinical heterogeneity (Rai K R, et al., Blood 46, 219, 1975; Binet
J L, et al., Cancer 48:198, 1981; and Rai K: A critical analysis of
staging in CLL, in Gail R, Rai K (eds): Chronic Lymphocytic
Leukemia. Recent Progress and Future Directions. New York, Alan R
Liss, 1987, p 253), they cannot accurately predict whether an early
or intermediate stage patient will experience an indolent or
aggressive course of disease. Specifically, since these systems
consider gross manifestations of the disease, including the level
of blood and marrow lymphocyte counts, the size and distribution of
the lymph nodes, the spleen size, the degree of anemia and the
patient's blood platelet count, they can only identify patients
with poor prognostic outcome when the disease has progressed to a
more advanced state.
At the present time, there is no known treatment for B-CLL which
has been shown to definitively increase life expectancy.
Consequently, only patients classified in the advanced stages of
B-CLL have been considered for aggressive treatment such as
chemotherapy, radiation therapy, surgery, immunotherapy or
transplantation. These treatments may exact a severe physical and
emotional toll on the patient without necessarily improving
outcome; in some instances, B-CLL patients may even succumb from
the rigors of treatment rather than from the effects of B-CLL.
Patients classified in the early stages of B-CLL, who may be in
better physical condition to receive more aggressive or
experimental treatment, generally receive no treatment as long as
the condition remains stable. This is for two reasons. First,
currently available therapies do not extend life span. Second,
there are currently no reliable indicators of which early stage
patients will do well and which will do poorly. Further, the
unpredictable course of the disease can make interpreting the
results of clinical trials difficult, as some early stage patients
will follow an indolent course even without the benefit of
treatment.
Such drawbacks have led researchers to develop adjuvant prognostic
criteria to be used in conjunction with the Rai and Binet staging
systems, including several parameters such as lymphocyte doubling
time (Montserrat E, et al., Br J Haematol 62:567, 1986),
circulating levels of .beta.2-microglobulin (Di Giovanni S, et al.,
Acta Haematol 81:181, 1989; Keating M J, et al., Blood 86:606A,
1995 (Abstract)), circulating levels of soluble CD23 (Sarfati M, et
al., Blood 88:4259, 1996), serum thymidine kinase levels (Kallander
C F, et al., Cancer 54:2450, 1984; Hallek M, et al., Blood 93:1732,
1999), bone marrow histology (Rozman C, et al., Blood 64:642,
1984), and cytogenetic abnormalities (Juliusson G, et al., N Engl J
Med 323:720, 1990).
An accurate prognostic indicator for B-CLL not related to the
symptoms of advanced disease would be desirable in the treatment
and case management of B-CLL patients. Specifically, a prognostic
indicator that could be evaluated at the cellular level at the
earliest stages of the disease (before onset of thrombocytopenia,
anemia, spleen and liver enlargement, etc.) would help physicians
identify which patients would progress to a more advanced state of
the disease and allow the option of more aggressive or experimental
treatment at a much earlier stage. Additionally, clinical trials of
new drugs or experimental therapies could be directed to patients
depending upon their prognostic outlook, thereby allowing for more
relevant results in clinical trials. Ideally, the expression of
such a prognostic indicator would remain constant over the course
of disease.
B-CLL is characterized by the clonal accumulation of CD5.sup.+
cells (Caligaris-Cappio, et al., J Exp Med 155:623-8, 1982).
Although these cells originally were considered antigen
inexperienced "virgin" lymphocytes, recent data indicate that at
least half of these cases represent expansions of
previously-triggered, post germinal center "memory" B cells
(Schroeder and Dighiero, Immunol Today 15:288-294, 1994; Fais, et
al., J Clin Invest 102:1515-1525, 1998). This conclusion is based
on the presence of significant numbers of somatic mutations in the
immunoglobulin (Ig) heavy (H) chain variable region (V) genes. In a
study of 83 (64 IgM.sup.+ and 19 non-IgM.sup.+) B-CLL cases, the
inventor and colleagues found significant numbers of V.sub.H
mutations in approximately 50% of the IgM.sup.+ and 75% of the
non-IgM+ (IgG and IgA) cases (Fais, et al, supra, 1998). Taken
together with newer studies undertaken by the inventor and
colleagues, V.sub.H and V.sub.L sequencing data suggest that
approximately 60% of B-CLL cases can be considered to be derived
from post-germinal center (GC) memory B-cells. Thus, the inventor
hypothesized that B-CLL cases can be divided into two categories,
namely cells clonally derived from post-germinal center memory
B-cells (hereinafter referred to as "post-GC B cells") and
pre-germinal center B cells (hereinafter referred to as "pre-GC B
cells"), some of which may be antigen inexperienced "virgin"
lymphocytes or activated B cells that were transformed without
entering a germinal center, and these categories may be relevant to
prognosis.
The expression of specific cell surface markers distinguishes
subsets of normal human B cells that differ in differentiation and
activation stages and in biologic properties (Clark and Lane, Ann
Rev Immunol 9:97-127, 1991). In particular, analyses of CD38 and
IgD expression have been especially useful in distinguishing
B-cells at various stages of differentiation from naive through
memory cells (Pascual, et al., J Exp Med 180:329-339, 1994; Zupo,
et al., Blood, 88:1365-1374, 1996).
Accordingly, the inventor sought to determine whether the
distinctions based upon surface membrane phenotype of B-CLL cells
(CD38.sup.+ or CD38.sup.-) or Ig V gene mutation status might
predict different clinical courses and outcomes for B-CLL patients
notwithstanding similar staging of these patients using
conventional staging methods. Undertaking the experiments described
herein, the inventor has discovered a strong correlation between
CD38 expression and Ig V gene mutation, and a strong independent
correlation between each of CD38 expression and IgV gene mutation
and patient prognosis. Since CD38 expression in a subject's B-CLL
cells may be easily and relatively inexpensively determined through
various methods known and commonly used in the art, CD38 expression
in particular may be a valuable prognostic indicator in B-CLL cases
and should aid in the management of B-CLL patients.
SUMMARY OF THE INVENTION
The present invention discloses a method for determining the
prognosis of a subject with chronic lymphocytic leukemia ("B-CLL"),
comprising determining whether the subject's B-CLL cells have been
clonally expanded from post-GC B cells (post-germinal center memory
B-cells) or pre-GC B cells (pre-germinal center B cells, some of
which may be antigen inexperienced "virgin" lymphocytes or
activated B cells that were transformed without entering a germinal
center), wherein clonal expansion from post-GC B cells may be
indicative of an indolent course of B-CLL in the subject or
favorable prognosis, and clonal expansion from pre-GC B cells may
be indicative of poor prognosis or an aggressive course of
disease.
In one method of the present invention, CD38 expression of B-CLL
cells in a biological sample from the subject is compared to a
baseline level of CD38 expression of B-CLL cells, wherein an
elevated level of CD38 expression in relation to the baseline level
of CD38 expression may indicate poor prognosis or aggressive course
of disease in the subject. In one embodiment, the percentage of
total B-CLL cells which are CD38.sup.+ in the biological sample is
compared to a baseline percentage of CD38.sup.+ B-CLL cells,
wherein an elevated percentage of CD38.sup.+ B-CLL cells in
relation to the baseline percentage of CD38.sup.+ B-CLL cells is
indicative of poor prognosis. In another embodiment of the
invention, the density of CD38 surface membrane expression on the
B-CLL cells in a biological sample from the subject is compared to
a baseline density of CD38 surface membrane expression of B-CLL
cells, wherein an elevated density of CD38 surface membrane
expression in relation to the baseline density of CD38 surface
membrane expression may indicate poor prognosis.
Also disclosed is a method for determining whether the Ig V genes
of the B-CLL cells of a B-CLL patient are mutated, comprising
comparing CD38 expression of B-CLL cells (either as a function of
relative percentage of CD38.sup.+ B-CLL cells, or as a function of
the relative density of CD38 surface membrane expression on the
B-CLL cells) in a biological sample from the subject to a baseline
level of CD38 expression, wherein a lower level of CD38 expression
in relation to the baseline level of CD38 expression indicates IG V
gene mutation.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts representative flow cytometric profiles of CD38
expression on mutated and unmutated CD5.sup.+/CD19.sup.+ B-CLL
cases. B-CLL cases were analyzed by flow cytometry after exposure
to anti-CD19-APC, anti-CD5-FITC, and anti-CD38-PE monoclonal
antibodies. The events illustrated were obtained by gating on cells
expressing CD19. Density plots of CD38 and CD5 expression are shown
for eight representative B-CLL cases. The upper four cases had no
mutations in either the V.sub.H or V.sub.L genes, whereas the lower
four cases had mutations in the V.sub.H and/or V.sub.L genes.
FIG. 2 illustrates the percentages of CD38-expressing B-CLL cells
among patients (n=37) whose Ig V.sub.H and V.sub.L genes had been
sequenced by the inventor and his colleagues. As established by
convention, unmutated cases (.diamond-solid.) were defined as cases
displaying <2.0% differences from the most similar germline
gene; mutated cases (.diamond.) display .gtoreq.2% differences.
Note that all the cases (17/17) that have .gtoreq.30% CD38.sup.+
B-CLL cells were unmutated, whereas only three unmutated cases
expressed low numbers (<30%) of CD38.sup.+ B-CLL cells. These
comparisons are statistically significant (p=0.00001; Mann-Whitney
test).
FIG. 3 depicts survival based on V gene mutation status and CD38
expression. Panel A is a Kaplan-Meier plot comparing survival based
on the absence ("unmutated": . . . ) or presence ("mutated": --) of
significant numbers (.gtoreq.2%) of V gene mutations in 47 B-CLL
cases (unmutated: 24 cases; mutated: 23). The median survival of
the unmutated group is 9 years; the median survival of the mutated
group was not reached in 19 years; p=0.0001; log-rank test). Panel
B is a Kaplan-Meier plot comparing survival based on the detection
of .gtoreq.30% ( . . . ) or <30% CD38.sup.+ B-CLL cells
(.gtoreq.30%: 17 cases; <30%: 19). The median survival of the
.gtoreq.30% CD38.sup.+ group is 10 years; the median survival of
the <30% CD38.sup.+ group was not reached in 19 years (p=0.0001;
log-rank test).
FIG. 4 depicts survival based on V gene mutation status and CD38
expression among B-CLL patients who stratify to the Rai
intermediate risk category. Panel A is a Kaplan-Meier plot
comparing V gene mutation status with survival among the cases
within the Rai intermediate risk category (unmutated: 16 cases;
mutated: 9). These cases are frustratingly difficult for clinicians
to treat because they can have either an indolent course requiring
no or minimal therapy, or a rapid downhill course despite
aggressive treatment.
The median survival of the mutated group is 17 years; the median
survival of the unmutated group is 9 years (p=0.0007; log-rank
test). Panel B is a Kaplan-Meier plot comparing numbers of
CD38.sup.+ B-CLL cells with survival among the cases within the Rai
intermediate risk category (.gtoreq.30%: 11 cases; <30%: 9
cases). The median survival of the 30% CD38.sup.+ group is 10
years; the median survival of the <30% CD38.sup.+ group was not
reached in 19 years (p=0.0030; log-rank test). None of the patients
in the <30% CD38.sup.+ group died during the follow-up
period.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for determining the
prognosis or projected clinical course in a subject with B cell
chronic lymphocytic leukemia ("B-CLL"). In particular, the method
of the present invention discloses an immunophenotypic prognostic
indicator which predicts whether the course of disease in a
specific B-CLL patient will be aggressive or indolent, thereby
aiding the clinician in managing the patient and evaluating the
modality of treatment to be used.
Since at the current time there are no known treatments that will
definitively increase the life expectancy of persons diagnosed with
B-CLL, clinicians must balance the rigors of aggressive or
experimental treatment with the likelihood that such treatment will
result in tangible benefit to the patient. In fact, some B-CLL
patients succumb to the combined effects of treatment and B-CLL
rather than to the effects of B-CLL alone. Accordingly, more
aggressive treatment, such as radiation therapy, chemotherapy,
transplants and immunotherapy, has traditionally been reserved for
those B-CLL patients already in the advanced stages of B-CLL who
stage higher in the conventional Rai and Binet staging systems.
However, these patients may be the most ill equipped to handle the
rigors of such treatment.
Additionally, the heterogeneous course of B-CLL complicates the
evaluation of clinical trials, as it is difficult to distinguish
patients who are effectively responding to the therapy being
administered from patients who would have never progressed to a
more advanced stage of the disease regardless of treatment.
Accordingly, an immunophenotypic prognostic indicator which is
predictive of a patient's clinical course, notwithstanding the
conventional stage of the disease, will aid clinicians in better
evaluating treatment options, as well as greatly enhancing the
value of clinical studies by better distinguishing the effects of
treatment.
The present invention generally disclose a method for determining
the prognosis of a subject with chronic lymphocytic leukemia
("B-CLL"), comprising determining whether the subject's B-CLL cells
(B-CLL B cells) have been clonally expanded from post-GC B cells
(post-germinal center memory B-cells) or pre-BC B cells
(pre-germinal center B cells, some of which may be antigen
inexperienced "virgin" lymphocytes or activated B cells that were
transformed without entering a germinal center). Clonal expansion
from post-GC B cells may be indicative of an indolent course of
B-CLL in the subject or favorable prognosis, while clonal expansion
from pre-GC B cells may be indicative of poor prognosis or an
aggressive course of disease.
Specifically, in the preferred embodiment of the present invention,
CD38 expression of B-CLL cells in a biological sample from the
subject is compared to a baseline level of CD38 expression of B-CLL
cells, wherein an elevated level of CD38 expression in relation to
the baseline level of CD38 expression may indicate poor prognosis
or aggressive course of disease in the subject. The method may be
performed using any tissue containing B-CLL cells, including but
not limited to spleen, lymph nodes, bone marrow, lymph, a whole
blood sample from the subject or a whole blood sample that has been
treated and processed to isolate the peripheral blood mononuclear
cells ("PBMC").
In one embodiment, the percentage of total B-CLL cells in the
biological sample which are CD38.sup.+ is compared to a baseline
percentage of CD38.sup.+ B-CLL cells, wherein an elevated
percentage of CD38.sup.+ B-CLL cells in relation to the baseline
percentage of CD38.sup.+ B-CLL cells is indicative of poor
prognosis, and a lower percentage of CD38.sup.+ B-CLL cells in
relation to the baseline percentage is indicative of a favorable
prognosis or indolent course of disease. Alternatively, the density
of CD38 surface membrane expression on the B-CLL cells in a
biological sample from the subject is compared to a baseline
density of CD38 surface membrane expression on B-CLL cells, wherein
an elevated density of CD38 surface membrane expression in relation
to the baseline density surface membrane expression may indicate
poor prognosis, and a lower density of CD38 surface membrane
expression in relation to the baseline density of CD38 surface
membrane expression may indicate a favorable prognosis or a more
indolent course of disease.
In both embodiments, i.e., comparing the relative percentage of
CD38.sup.+ B-CLL cells (as a percentage of the B-CLL population in
total, such as a percentage of CD5.sup.+/CD19.sup.+ lymphocytes) or
comparing the relative density of CD38 surface membrane expression
on B-CLL cells in the biological sample, the level of CD38
expression may be determined by any method currently known in the
art, including any applicable direct or indirect immunofluorescence
technique. Further, where relative density of CD38 surface membrane
expression on the B-CLL cells in the biological sample is being
determined, mean channel fluorescence may be used. In a preferred
embodiment of the invention, the level of CD38.sup.+ B-CLL
expression is determined using flow cytometry where the cells have
been labeled with monoclonal antibodies conjugated with fluorescent
dyes or enzymes, although visual immunofluorescence or other
methods may also be used. In a specific embodiment, PBMCs are
analyzed for surface expression of CD19/CD5/CD38 by triple color
immunofluorescence using anti-CD19-APC, anti-CD5-FITC (CD5 being
specific to B-CLL cells and CD19 being specific to B lymphocytes,
although other combinations of antigens/labeled antibodies or
enzymes may be used that are specific to narrow the analyzed pool
to B-CLL cells) and anti-CD38-PE antibody conjugates. The preferred
antibody conjugate is anti-CD38-PE (Simultest LeucoGATE, from
Becton Dickinson Immunocytometry Systems, San Jose, Calif.).
Additionally, it is well within the skill of one of ordinary skill
in the art to devise either direct or indirect immunoassay kits
(i.e., ELISA or other kits) which use similar antigen/labeled
antibody or enzyme combinations to detect levels of CD38
expression. The relative percentage of CD38.sup.+ B-CLL cells in
relation to a percentage baseline of CD38.sup.+ B-CLL cells, or the
relative density of CD38 surface membrane expression on B-CLL cells
in relation to a baseline density of CD38 surface membrane
expression, may be determined by comparing the resulting color,
fluorescence or equivalent reaction with a control sample having a
predetermined percentage of CD38.sup.+ B-CLL cells or density of
CD38 (or the relevant epitope of CD38), as appropriate.
While the exact relative percentage of CD38.sup.+ B-CLL cells or
density of CD38 surface membrane expression that defines poor or
favorable prognosis, i.e., the baseline level of CD38 expression of
the B-CLL cells, is somewhat arbitrary (as the numerical cut off
value may be shifted upward or downward with an attendant loss of
accuracy in the prognostic utility of the test), in a preferred
embodiment of the invention using the disclosed antibody or one of
equivalent avidity and specificity, as well as the disclosed
anti-CD38-PE fluorochrome, the relative percentage of CD38.sup.+
B-CLL cells indicating poor prognosis is greater than 15%, more
preferably is greater than 20%, even more preferably is greater
than 25% and most preferably is greater than 30% of the total B-CLL
cells in the biological sample. The preferred range may be affected
by the specific anti-CD38 monoclonal antibody (mAB) used, as
different mABs will have different binding affinities (avidity) for
CD38 and may bind to different epitopes of CD38, as well as by the
specific fluorochrome used. Further, similar variable parameters
exist in isolating the B-CLL population in the biological sample.
That is, the exact preferred baseline may vary depending upon the
specific anti-CD5 and anti-CD19 mABs used, and the anti-CD5 and
anti-CD19 fluorochrome conjugates used, for the reasons noted above
regarding variations in avidity and specificity, as well as the
efficiency of the mAB conjugated fluorochrome. Using the mABs and
fluorochromes disclosed in the Experimental Details below will
yield the preferred baseline range of CD38 expression as disclosed
herein. However, all other elements being the same, mABs with a
lower avidity will have a correspondingly increased baseline range,
so that higher densities of CD38 surface membrane expression, or
increased relative percentages of CD38.sup.+ B-CLL cells, will be
required to establish a poor prognosis or aggressive course of
disease.
However, it is well within the skill of one of ordinary skill in
the art to determine the appropriate CD38 baseline level, by either
using the experimental method disclosed herein (that is, comparing
levels of CD38 expression among a characterized group of B-CLL
patients with a known clinical course), or by comparing the
relative avidity and specificity of the mABs disclosed herein and
the mABs used in any particular instance, as well as the relative
efficiency of the particular fluorochrome used, and thereafter
deducing the appropriate baseline.
Also disclosed by the present invention is a method for determining
whether the Ig V genes of the B-CLL cells of a B-CLL patient are
mutated, comprising comparing CD38 expression of B-CLL cells in a
biological sample from the subject to a baseline level of CD38
expression of B-CLL cells, wherein a lower level of CD38 expression
in relation to the baseline level of CD38 expression indicates IG V
gene mutation. In one embodiment of the invention, CD38 expression
of B-CLL cells in the biological sample is compared to the baseline
level of CD38 expression by comparing the percentage of total B-CLL
cells which are CD38.sup.+ to a baseline percentage of CD38.sup.+
B-CLL cells, wherein a lower percentage of CD38.sup.+ B-CLL cells
in relation to the baseline percentage is indicative of mutated IgV
genes of the B-CLL cells. Preferably, the percentage of CD38.sup.+
B-CLL cells indicating Ig V gene mutation is 15% or less, more
preferably is 20% or less, even more preferably is 25% or less, and
most preferably is 30% or less. In another embodiment of the
invention, CD38 expression of the B-CLL cells in the biological
sample is compared to the baseline level of CD38 expression by
comparing the density of CD38 surface membrane expression of the
B-CLL cells in relation to a baseline of CD38 surface membrane
expression, wherein a lower density of CD38 surface membrane
expression in relation to the baseline is indicative of mutated IgV
genes of the B-CLL cells.
The present invention is described in the following Experimental
Details Section which is set forth to aid in the understanding of
the invention, and should not be construed to limit in any way the
invention as defined in the claims which follow thereafter.
Experimental Details Section
Methods
(i) Patients
The Institutional Review Boards of North Shore University Hospital,
Manhasset, N.Y., and Long Island Jewish Medical Center, New Hyde
Park, N.Y., approved these studies. The patients in this study are
a subset (n=47) of the well-defined cohort (n=64) of randomly
chosen, typical IgM.sup.+ B-CLL patients described previously (Fais
F, et al., J Clin Invest 102:1515, 1998). Patients were selected
for the present study based on the availability of detailed
clinical histories (Don Monti Division of Medical Oncology, North
Shore University Hospital, and the Hematology/Oncology Division,
Long Island Jewish Medical Center) and the availability of DNA
sequences for both the Ig V.sub.H (Fais F, et al., supra, J Clin
Invest 102:1515, 1998) and V.sub.L (Ghiotto et al., in preparation)
genes in each case. The clinical courses of the patients that were
analyzed in this study were not significantly different from those
that could not be studied because of lack of sample or follow-up.
There were 34 males and 13 females in this group, with a mean age
of 63.4 years (range: 38-80). The mean ages of the unmutated (mean:
61.3; range 38-79) and mutated (mean: 65.5; range: 47-80) cases or
of the .gtoreq.30% CD38.sup.+ (mean: 63.5; range 38-79) and <30%
CD38.sup.+ (mean: 63.6; range 44-79) cases were similar. Fresh or
cryopreserved B-CLL cells were available for surface marker
analyses on 37 patients (20 unmutated and 17 mutated). These
samples had been obtained at various points in the clinical
follow-up of these patients. There were no differences in the
timing of sample acquisition between the unmutated and mutated
groups.
(ii) Cellular Immunophenotypic Analysis
The following antibody conjugates were used: anti-CD23-fluorescein
isothiocyanate (FITC; Immunotech, Inc., Westbrook, Me.), goat
anti-human IgD-FITC (Southern Biotechnology Associates, Birmingham,
Ala.), and anti-CD5-FITC, anti-CD5-phycoerythrin (PE),
anti-CD38-PE, anti-CD19-allophycocyanin, anti-CD45-FITC and
anti-CD14-PE (Simultest LeucoGATE; all from Becton Dickinson
Immunocytometry Systems, San Jose, Calif.).
Peripheral blood mononuclear cells (PBMC) were separated from
heparinized venous blood by density gradient centrifugation using
Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, N.J.), and
used either immediately or after thawing samples that had been
cryopreserved with a programmable cell freezing machine (CryoMed,
Mt. Clemens, Mich.). PBMCs were analyzed for surface expression of
CD19/CD5/CD38 and CD19/IgD/CD38 and CD19/CD5/CD23 by triple color
immunofluorescence (Fais F, et al., J Clin Invest 98:1659, 1996).
For the detection of CD19/CD5/CD38-expressing cells, mAb labeled
with the following fluorochrome were used: anti-CD19-APC,
anti-CD5-FITC, and anti-CD38-PE; for the detection of
CD19/IgD/CD38-expressing cells, anti-CD19-APC, anti-IgD-FITC, and
anti-CD38-PE mAb were used; for the detection of
CD19/CD5/CD23-expressing cells, anti-CD19-APC, anti-CD23-FITC, and
anti-CD5-PE mAb were used. Isotype-matched negative controls were
used in all assays to determine positive from negative results.
Flow cytometric analyses were performed on a Becton-Dickinson FACS
Calibur flow cytometer equipped with argon and red diode lasers.
Measurements of forward and side scatter were combined with CD45
and CD14 determinations to identify lymphocytes and exclude
monocytes. The CellQuest software system was used to acquire and
analyze data.
(iii) Preparation of RNA and cDNA Synthesis
Total RNA was isolated from either fresh or cryopreserved B-CLL
cells using Ultraspec RNA (Biotecx Laboratories, Houston, Tex.)
according to the manufacturer's instructions. Two .mu.g of RNA were
reverse transcribed to cDNA using M-MLV reverse transcriptase
(GIBCO BRL, Life Technologies, Grand Island, N.Y.) and 20 pmol of
oligo dT primer in a total volume of 20 .mu.l (Fais, et al., J.
Clin. Invest., 102(8), 1998, 1515-1525). These reactions were
carried out at 42.degree. C. for 1 hr, heated at 65.degree. C. for
10 minutes, and then diluted to a final volume of 100 .mu.l.
To determine the sequence of the B-CLL cells, 3 .mu.l of cDNA were
amplified using a sense V.sub.H leader family specific primer in
conjunction with an antisense 19mer C.sub.H primer. The reaction
was carried out in 50 .mu.l using 20 pm of each primer and cycled
with a Perkin Elmer Cetus 9600 apparatus (Emeryville, Calif.) as
follows: denaturation at 94.degree. C. for 30 sec.; annealing at
55.degree. C. for 30 sec.; and extension at 72.degree. C. for 1
min. After 35 cycles, extension was continued at 72.degree. C. for
an additional 10 min.
The V.sub.H PCR product was either sequenced in both directions by
the dideoxy-chain termination method using fluorescent-labeled
ddNTP and TAQ polymerase (Applied Biosystems, I System, Foster
City, Calif.) and an automated sequencer (Applied Biosystems,
Foster City, Calif.). Sequencing was performed either directly
after purification of PCR products with Wizard PCR Preps (Promega,
Madison, Wis.), or after cloning into TA vector (Invitrogen, San
Diego, Calif.). When the cloning approach was used, multiple
colonies were chosen randomly and sequenced.
The B-CLL sequences obtained were compared with those in the V BASE
directory using MacVector software, version 6.0 (Eastman Kodak Co.,
New Haven, Conn.) to determine the most similar germline VH gene
and the degree of difference from this germline gene. Similar
methods were used to obtain the V.sub.L gene sequences and
determine the degree of difference from the germ line gene.
However, V.sub.L genes were amplified using a sense V.sub.L leader
family specific primer in conjunction with an antisense C.sub.L
primer.
(iv) Statistical Analyses
The percentages of CD5.sup.+/CD19.sup.+ B cells that co-expressed
CD38 or IgD or CD23 were determined for each patient and
statistical differences between the unmutated and mutated groups
analyzed using the Mann-Whitney test. Patients were also classified
according to the percentage of B-CLL cells expressing CD38 into
.gtoreq.30% CD38.sup.+ and <30% CD38.sup.+ groups.
To determine the degree of association between the individual
patients based on the actual percentages of CD38-expressing cells
and on the V gene mutation status or on the percentages of
CD23-expressing cells, the Spearman coefficient was calculated. To
determine the degree of association between the patients classified
into two groups based on percentages of CD38-expressing cells and
on the V gene mutation status, the Kappa coefficient was used.
Standard methods for estimating proportions and associated exact
confidence intervals (CI) were used for estimating sensitivity of
high numbers of CD38.sup.+ B-CLL cells (.gtoreq.30%) as a marker
for "unmutated" V genes. In standard epidemiological terminology,
the unmutated gene corresponded to the "disease" state and
accordingly sensitivity was computed using the number of patients
with unmutated genes as the denominator. Similarly, specificity of
low numbers of CD38.sup.+ B-CLL cells (<30%) was computed using
the patients with mutated genes as the denominator. Accuracy was
defined as the percentage of patients who were classified correctly
as unmutated or mutated using the CD38 criteria. Positive and
negative predictive values were computed using Bayes' Rule.
Comparisons of V gene mutation status and CD38 expression with
clinical course were made "blindly". The investigator who reviewed
the clinical histories of these patients was unaware of the
laboratory data during the retrospective chart review. The
two-tailed Fisher's Exact test was used to determine whether
chemotherapy requirements, Rai stage at diagnosis, or gender were
significantly associated with V gene mutation status or with CD38
percentage. Survival analyses were performed using the Kaplan-Meier
product-limit method and the log-rank test.
Results
(i) Percentages of CD38.sup.+ B-CLL Cells Among the Unmutated and
Mutated Cases
The DNA sequences of the Ig V.sub.H (Fais F, et al, supra, J Clin
Invest 102:1515, 1998) and V.sub.L (Ghiotto et al., in preparation)
genes expressed by the leukemic cells of the 47 typical IgM.sup.+
B-CLL cases included in this study were determined previously.
Based on the numbers of somatic mutations detected in these genes,
the cases were divided into two categories: "unmutated" or
"mutated". As per current convention, "unmutated" cases were
defined as those with <2% differences from the most similar
germline gene in both the expressed V.sub.H and V.sub.L genes;
"mutated" cases were defined as those in which the B-CLL cells
displayed .gtoreq.2% differences in either the expressed V.sub.H or
V.sub.L gene.
To determine whether these genetic differences reflected cellular
phenotypic differences, the expression of CD38 and IgD on the B-CLL
cells of the 37 patients in whom PBMC were available (20 unmutated
and 17 mutated) were analyzed. Analyses of CD38 and IgD expression
were chosen for these studies because they distinguish B cells at
various stages of differentiation (Clark E A, and Lane P J, Annu
Rev Immunol 9:97, 1991; Pascual V, et al, J Exp Med 180:329,
1994).
The unmutated and mutated B-CLL cases were similar in CD19.sup.+ B
cells co-expressing CD5, CD23 and IgD, both in the percentages of
positive cells and in mean fluorescence intensity (data not shown).
However, the percentage of CD38.sup.+ cells was dramatically
different between the unmutated and mutated cases. FIG. 1
illustrates eight representative B-CLL cases analyzed for
CD19/CD5/CD38-expressing cells. The V.sub.H and V.sub.L genes of
the four cases listed in the upper panel were not mutated, whereas
the V.sub.H and/or V.sub.L genes in the lower panel were mutated.
Note that the unmutated cases have a much higher percentage of
CD38.sup.+ cells among the CD5.sup.+/CD19.sup.+ cells than the
mutated cases in the lower panel.
When the percentages of CD38.sup.+ B-CLL cells in the unmutated and
mutated groups were compared statistically, very significant
differences were found (means: 63.9% vs. 7.3%, respectively;
p=0.00001). The Spearman correlation between the individual
percentages of CD38.sup.+ B-CLL cells in each case vs the actual
percentages of V gene mutations was r=-0.75 (p<0.001),
indicating a relatively strong inverse relationship. Accordingly,
while CD38 is clearly an independent marker for predicting clinical
outlook, the statistically significant inverse relationship between
CD38 expression and V gene mutation indicates that measurements of
CD38 expression can be used to evaluate the level of V gene
mutation. A low to moderate direct correlation existed between the
percentages of CD23.sup.+ B-CLL cells and the percentage of V gene
mutation r=0.42; p=0.01). There was no correlation between CD38
expression and CD23 expression (data not shown).
When the results on percentages of CD38 expressing cells were
plotted individually (FIG. 2), the cases could be segregated into
two distinct sets, one with .gtoreq.30% CD38.sup.+ cells and the
other with <30% CD38.sup.+ cells. The 30% cut off value was
chosen empirically based on the observed distributions on the plot,
and it is understood that higher or lower cut off value may be
chosen though such a cut off point may result in lower accuracy in
determining prognosis. Furthermore, an inverse relationship existed
between CD38 expression and V gene mutation status. The set with
the higher percentages of CD38.sup.+ B-CLL cells was comprised
solely of unmutated cases, whereas the set with the lower
percentages of CD38.sup.+ cells contained all of the mutated cases
and three of the unmutated cases. The Kappa coefficient calculated
for association between these two sets of CD38.sup.+ B-CLL cases
vs. the unmutated and mutated groups was -0.84, indicating a strong
inverse relationship.
Furthermore, high percentages of CD38.sup.+ B cells (.gtoreq.30%)
indicated the presence of <2% mutations with 100% specificity
(95% C.I.: 84-100%). Since three patients with unmutated V genes
were found to have <30% CD38.sup.+ B-CLL cells (FIG. 2), the
sensitivity of using .gtoreq.30% CD38.sup.+ B-CLL cells as a marker
for significant percentages of V.sub.H or V.sub.L gene mutations
was 85% (95% C.I.: 62-97%). Based on this specificity and
sensitivity and on a prevalence of 60% for .gtoreq.2% mutations in
either V.sub.H or V.sub.L, the positive predictive value of
.gtoreq.30% CD38.sup.+ B cells indicating the "unmutated" genotype
was 100%. Conversely, the predictive value of <30% CD38.sup.+ B
cells indicating the "mutated" genotype was 82%. These CD38
criteria indicate V gene mutation status with 92% accuracy.
The differences in CD38 expression were stable over time and were
not influenced by chemotherapy. Sixteen patients (7 with CD38
values .gtoreq.30% and 9 with <30%) were studied at two or more
time points, separated by as much as 6 years. Indeed, the
percentages of CD38.sup.+ B-CLL cells detected never differed by
more than 10% in any instance. One patient with 95% circulating
CD38.sup.+ B-CLL cells was studied on five occasions over a 24
month period and the percentages of CD38.sup.+ cells detected in
each sample were very similar (<5% difference).
(ii) Clinical Course and Outcome of the Unmutated vs. Mutated B-CLL
Cases
The treatment histories of the patients with unmutated and mutated
Ig V region genes were very different (Tbl. 1). Eighteen of the 23
mutated cases (78.3%) required either no chemotherapy (52.2%) or
minimal treatment (26.1%), while only 20.8% of the unmutated cases
required no (16.6%) or minimal therapy (4.2%). These differences
were highly significant (p=0.0001). Furthermore, 79.2% (19/24) of
the unmutated cases required continuous chemotherapy or
chemotherapy utilizing two or more agents or regimens. Although 18
of these 19 patients (94.7%) received fludarabine, only two
achieved a durable clinical response.
TABLE-US-00001 TABLE 1 Comparison of Treatment Histories Based on
Either Ig V Gene Mutation Status or the Percentages of CD38.sup.+
B-CLL Cells Treatment Unmutated Mutated Patients requiring no or
minimal 20.8% (5/24) 78.3% (18/23) treatment* Patients requiring
continuous 79.2% (19/24) 21.7% (5/23) chemotherapy or chemotherapy
with 2 or more agents or regimens p = 0.0001.dagger. .gtoreq.30%
CD38+ <30% CD38+ Treatment B-CLL Cells B-CLL Cells Patients
requiring no or minimal 23.5% (4/17) 73.7% (14/19) treatment*
Patients requiring continuous 76.5% (13/17) 26.3% (5/19)
chemotherapy or chemotherapy with 2 or more agents or regimens p =
0.0067.dagger. *Minimal treatment is defined as less than 6 months
of therapy in the years of follow up. .dagger.Statistical analyses
performed using the two-tailed Fisher's exact test.
These significant differences in chemotherapy requirements were
reflected in significant differences in survival (FIG. 3A). The
median survival of the patients in the unmutated group was 9 years,
whereas median survival for the mutated group was not reached for
the duration of follow-up (p=0.0001).
Finally, V gene mutation status was compared with the clinical
stage at the time of diagnosis using the modified Rai system. The
patients stratified to all Rai modified clinical stages at the time
of diagnosis (Tbl. 2). Patients who stratify to the Rai
intermediate risk group are the most heterogeneous in treatment
requirements and survival and represent those in whom outcome is
the most difficult to predict (Rai K, et al., supra, in Hematology:
Basic Principles and Practice (ed 2.sup.nd), p 1308; Zwiebel and
Cheson, Semin Oncol, 25:42-59, 1998). Therefore, the survival of 25
patients in this group were analyzed (9 mutated cases vs. 16
unmutated cases; FIG. 4A). The median survival of the unmutated
cases was 9 years, compared to 17 years for the mutated cases
(p=0.0007).
TABLE-US-00002 TABLE 2 Comparison of Modified Rai Stage at
Diagnosis with Ig V Gene Mutation Status and the Percentages of
CD38.sup.+ B-CLL Cells Stage Unmutated Mutated Low* 22.7% (5/22)
52.4% (11/21) Intermediate 72.7% (16/22) 42.9% (9/21) High 4.6%
(1/22) 4.7% (1/21) p = 0.123 <30% CD38+ B-CLL .gtoreq.30% CD38+
B-CLL Cells Cells Low .dagger. 20.0% (3/15) 50.0% (9/18)
Intermediate .dagger. 73.3% (11/15) 50.0% (9/18) High 6.7% (1/15)
0.0% (0/18) p = 0.138 *Comparison of V gene mutation status among
patients in the low and intermediate risk categories (p = 0.058;
two-tailed Fisher's Exact test). .dagger. Comparison of CD38
expression among patients in the low and intermediate risk
categories (p = 0.147; two-tailed Fisher's Exact test).
(iii) Clinical Course and Outcome of B-CLL Cases with .gtoreq.30%
or <30% CD38.sup.+ Cells.
Since there was a significant correlation between V gene mutation
and CD38 expression by the B-CLL cells, chemotherapy requirements
and survival as a function of the percentages of CD38.sup.+
leukemic cells were compared. Significant differences were found
for both. Seventy three percent (14/19) of the <30% CD38.sup.+
cases required either no or minimal chemotherapy, compared with
23.5% (4/17) of the .gtoreq.30% CD38.sup.+ cases (p=0.0067; Tbl.
1). Conversely, 76.5% of the .gtoreq.30% CD38.sup.+ cases required
either continuous chemotherapy or chemotherapy with two or more
agents or regimens.
Median survival for the patients in the .gtoreq.30% CD38.sup.+
group was 10 years (FIG. 3B). In contrast, this value could not be
determined for the patients in the <30% CD38.sup.+ group since
all patients in this group were alive for the duration of follow-up
(p=0.0001). Highly significant differences in survival also were
found among the patients in the Rai intermediate risk group (FIG.
4B). Median survival for the .gtoreq.30% CD38.sup.+ patients was
reached in 10 years, whereas all patients in the <30% CD38.sup.+
group remained alive throughout the years of follow-up
(p=0.003).
(iv) Studies of IgG.sup.+ and IgA.sup.+ B-CLL Cases.
The preceding observations were also true for a cohort of non-IgM
producing (IgG or IgA) B-CLL patients (n=16), whose V gene sequence
analyses were published previously (Fais, et al., supra, J Clin
Invest 102:1515-25, 1998; Hashimoto, et al., supra, J Exp Med
181:1507-17, 1995). The median survival of the unmutated non-IgM
cases was only 3 years, whereas it was not reached for the mutated
cases at 15 years (p=0.004, log-rank test; data not shown). Similar
data were obtained when the cases were compared based on CD38
expression, although the small numbers of available samples (n=8)
precluded accurate statistical analysis. When these non-IgM.sup.+
cases were pooled with the IgM.sup.+ cases described above
(bringing the total number of patients studied to 63), the median
survival for the unmutated group (n=29) was 8 years and for the
mutated group (n=34) was not reached for the duration of follow-up
(p=0.0001). Similar data were obtained for the CD38 groups: median
survival for the .gtoreq.30% CD38.sup.+ (n=19) was 9 years, whereas
median survival for the <30% CD38.sup.+ group (n=25) was not
reached (p=0.0001).
(v) Gender of the B-CLL Cases Based on Either V Gene Mutation or
CD38 Expression
The cohort of IgM.sup.+ B-CLL patients in this study consisted of
34 males and 13 females (M:F=2.6:1). However, the M:F ratio of the
patients stratified by either V gene mutation status or CD38
expression was very different (Tbl. 3).
In the mutated group, males and females were virtually equally
distributed, whereas in the unmutated group a marked male
predominance was found (M:F ratio=11:1; p=0.003). A similar
disparity in gender distribution was seen when the patients were
compared based on the percentages of CD38.sup.+ B-CLL cells. The
numbers of males and females among the <30% CD38.sup.+ group
were almost equal (M:F=1.1:1), whereas males outnumbered females in
the .gtoreq.30% CD38.sup.+ group (M:F=7.5:1; p=0.031).
TABLE-US-00003 TABLE 3 Gender Differences Based on either Ig V Gene
Mutation Status or the Percentages of CD38.sup.+ B-CLL Cells
Unmutated Mutated Male* 91.7% (22/24) 52.2% (12/23) Female* 8.3%
(2/24) 47.8% (11/23) Male:Female Ratio 11.0:1 1.1:1 p = 0.003*
.gtoreq.30% CD38+ B-CLL <30% CD38+ B-CLL Cells Cells Male* 88.2%
(15/17) 52.6% (10/19) Female * 11.8% (2/17) 47.4% (9/19)
Male:Female Ratio 7.5:1 1.1:1 p = 0.031* *Statistically significant
difference in gender distribution between the unmutated and mutated
groups and between the .gtoreq.30% CD38.sup.+ and <30%
CD38.sup.+ groups (two-tailed Fisher's Exact test).
TABLE-US-00004 TABLE 4 Characteristics of the Two Groups of B-CLL
Patients % of CD38+ Chemotherapy M:F V Gene Status B-CLL Cells
Requirement Survival Ratio Group 1 Unmutated High Extensive Shorter
High Group 2 Mutated Low Minimal Longer ~ Equal
Discussion
The preceding data indicate that Ig V gene mutation status and CD38
expression are both distinct and important prognostic indicators of
clinical course and outcome in B-CLL. Indeed, those patients in
either the unmutated or .gtoreq.30% CD38.sup.+ groups experienced a
worse clinical course than those patients in the mutated or <30%
CD38.sup.+ groups. This was true for both chemotherapy requirements
(Tbl. 1) and survival (FIG. 3).
Possibly the most clinically relevant correlation was found among
those patients who presented initially in the Rai intermediate risk
category (FIG. 4).
These patients are frustratingly difficult for clinicians to treat
because they can have either an indolent course requiring no or
minimal therapy or a rapid downhill course despite aggressive
treatment. Both CD38 expression and V gene mutation status were
able to segregate those Rai intermediate risk patients who followed
an indolent course from those whose course was much more aggressive
(FIG. 4).
Relevant to observations on Ig V gene mutations and survival is the
study of Oscier et al (Blood 89:4153-60, 1997), indicating that
B-CLL cells with unmutated V.sub.H genes frequently contain three
copies of chromosome 12, a cytogenetic marker that is associated
with poor clinical outcome. This study was recently extended by
Hamblin et al (Blood, 1999 Sep 15;94(6):1848-54) to a larger cohort
of patients. These new data are consistent with the observations
herein and show clearly that unmutated V.sub.H genes are associated
with a more aggressive form of B-CLL.
When the subject patients were stratified according to V gene
mutation status or CD38 expression (Tbl. 3), a clear preponderance
of males was noted in the unmutated and .gtoreq.30% CD38.sup.+
(poor outcome) groups (M:F: 11:1 and 7.5:1, respectively). These
ratios are much higher than those reported previously (Catovsky, et
al., Br J Haematol 72:141-9, 1989). The subject data, however,
agree with the studies (Catovsky, et al, supra; Mandelli, et al.,
supra, J Clin Oncol 5:398-406, 1987) indicating that women with
B-CLL have a more favorable clinical course than men. Although
women comprised only .about.10% of the unmutated and .gtoreq.30%
CD38.sup.+ (poor outcome) groups, they constituted .about.50% of
the mutated and <30% CD38.sup.+ (good outcome) groups (Tbl. 3).
These data support a role for gender indirectly influencing
clinical outcome and possibly B cell maturation and
differentiation. The mechanism(s) responsible for these differences
are obscure at this point.
The two sets of B-CLL cases characterized in this study appear to
represent B cells transformed at different stages of B cell
differentiation and/or activation. Thus, those B-CLL cases with
mutated V genes and low numbers of CD38.sup.+ B-CLL cells are
characteristic of post-GC, memory B cells (Clark, Annu Rev Immunol
9:97-127, 1991; Pascual, et al, J Exp Med 180:329-39, 1994). Some
of these B-CLL cells may be derived from the small subset of
IgM.sup.+/IgD.sup.+ memory cells found in the blood (Klein, et al,
J Exp Med 188:1679-89, 1998) or bone marrow (Paramithiotis and
Cooper, Proc Natl Acad Sci USA 94:208-212, 1997) or from cells
similar to the IgD.sup.+ memory B cells identified in tonsils
(Arpin, et al., J Exp Med 187:1169-78, 1998). Although CD27 is
another marker that distinguishes pre-GC from post-GC B cells
(Klein, et al, supra, J Exp Med 188:1679-89, 1998; Agematsu, et
al., J Immunol 153:1421-9, 1994; Tangye, et al., J Exp Med
188:1691-703, 1998), differences in CD27 expression among the
subject CD5.sup.+ B-CLL cases were not found, either in density per
cell or in cell number (data not shown). These data are in
agreement with those of others (Ranheim, et al, Blood 85:3556-65,
1995; Trentin, et al., Cancer Res 57:4940-7, 1997).
In contrast, those B-CLL cases with unmutated V genes and high
numbers of CD38.sup.+ B-CLL cells display surface markers
characteristic of B cells that have not entered a GC. Since CD38,
as detected by mAb conjugated with PE (phycoerythrin), is expressed
on most blood B cells (Kumagai, J Exp Med 181:1101-10, 1995), the
.gtoreq.30% CD38.sup.+/unmutated B-CLL cells could be derived from
either naive B cells or activated B cells that have not entered a
GC and have not generated Ig V gene mutations. Based on analyses of
the HCDR3 characteristics of unmutated B-CLL cases (Fais, et al,
supra, J Clin Invest 102:1515-25, 1998; Johnson, et al., J Immunol
158:235-46, 1997), a favored hypothesis is that some of these
unmutated B-CLL cells have been activated and selected by
antigen.
The physiological significance of CD38 expression primarily by the
unmutated cases and its potential function in cell survival and
proliferation is presently unknown. However, previous studies
suggest that CD38 expression identifies those B-CLL clones that are
capable of transducing signals through their B cell antigen
receptors that may increase or decrease their chance for survival
(Lam, et al., Cell 90:1073-83, 1997; Zupo, et al., Eur J Immunol
24:1218-1222, 1994). In this regard, Zupo et al have reported that
CD38.sup.+ B-CLL cells can be induced to undergo apoptosis in vitro
after exposure to anti-Ig antibodies, whereas CD38.sup.- B-CLL
cells are resistant to these effects. These data are at variance
with the unexpected subject clinical observations that those B-CLL
cases with higher percentages of CD38.sup.+ B cells have a worse
clinical outcome. However, it is possible that the quality of the
antigen receptor stimulus and the presence of associated stimuli
may lead to diverse endpoints (apoptosis vs. survival). Similarly,
triggering through the CD38 molecule can have different effects on
the survival of B cells depending on the state of
maturation/activation of the cell. Whereas anti-CD38-mediated
signaling results in the death of immature B cells (Kumagai, supra,
J Exp Med 181:1101-1-, 1995), mature B cells can be rescued from
apoptosis by CD38 triggering (Lam, et al., supra, Cell 90:1073-83,
1997; Zupo, et al, supra, Eur J Immunol 24:1218-1222, 1994).
Further studies will be necessary to determine how these in vitro
data correlate with the subject clinical observations.
In conclusion, the present studies identify CD38 expression and V
gene mutation status as novel and independent prognostic indicators
that appear to identify mutually overlapping groups of B-CLL
patients (FIGS. 1 and 2 and Results). However, since CD38
expression can be determined more conveniently and rapidly than Ig
V gene mutations, this parameter may be the preferred adjunct to
the current staging systems.
Indeed, the results of this simple test should enable physicians to
predict with considerable accuracy whether a patient is likely to
have a favorable or unfavorable clinical course. Furthermore, since
the leukemic cells appear to be fixed in their level of expression
of this marker, determining the percentage of CD38.sup.+ B-CLL
cells may be useful at any point in the clinical course of the
individual B-CLL patient. However, the possibility that CD38
expression might change with the alterations in chromosomal
structure and gene expression that occur in Richter's
transformation cannot be excluded (Koduru, et al., Br J Haematol
85:613-616, 1993).
All publications mentioned herein above are hereby incorporated by
reference in their entirety. While the foregoing invention has been
described in some detail for purposes of clarity and understanding,
it will be appreciated by one skilled in the art from a reading of
the disclosure that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
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